ABSTRACT Irritable bowel syndrome (IBS) is associated with colonic motor, sensory, mucosal and psychological dys- functions. The etiology of IBS with diarrhea (IBS-D) includes exogenous factors (e.g. stress or infections) and endogenous molecules [e.g. bile acids (BA) and short chain fatty acids (SCFA)] that increase 5-HT release, mucosal permeability, low grade inflammation, and colonic secretion and motility. There are univariate genetic associations of single nucleotide polymorphisms (SNPs in 5-HTTLPR, 5-HT3R, FAAH, CNR1, KLB, FGFR4, TGR5, TLR9, CDH1, TNFSF15, IL6, NXPH1, KDELR2 , GRID2IP, NPSR1, HLA DQ2/8) with IBS phenotype (including GWAS) or quantitative traits. We previously examined the association of BA in IBS-D traits with: colonic transit, sensation, permeability, BA synthesis and excretion. On meta-analysis,~28% of patients IBS-D have BA diarrhea, reduced secretion of the ileal hormone FGF-19, and univariate association with 3 BA- related genes: KLB, FGFR4, and TGR5. Overall aim of this proposal is to understand mechanisms in IBS-D, especially in IBS-D patients with increased BA excretion. Preliminary data explored exome DNA sequencing, and colonic mucosal mRNA expression in IBS-D: (i). Our exome DNA sequencing study (16 IBS-D, 50 controls) confirmed SNPs in FGFR4 and KLB are associated with colonic transit and BA homeostasis, and identified new single nucleotide variants (SNVs) in FGFR4 and KLB in IBS-D. However, the relationship of the SNVs to IBS-D quantitative traits is unknown. (ii).Our first-ever RNA-Seq study of rectosigmoid mucosa (9 IBS-D and 9 controls) showed 4 pathways with increased expression of neurotransmitters or their receptors (P2RY4, VIP), ion transport (GUC2AB, PDZD3), cytokines (C4BPA), BA modulation (FGFR4), and decreased expression of barrier mechanisms (FN1). (iii). In 47 IBS-D patients, we confirmed mRNA changes in ion transport, immune and barrier functions. (iv). Open-label treatment with BA sequestrant, normalized stool consistency in IBS-D with increased fecal BA. Our data demonstrate feasibility to use deep phenotyping, next generation sequencing of 5 BA-related genes, and colonic mucosal mRNA expression to identify mechanism in IBS-D. With unique capability to perform deep phenotyping of IBS-D (transit, sensation, permeability, mucosal barrier, BA kinetics, fecal SCFA profile, mucosal transcriptome and proteomics), we aim to test 3 hypotheses: 1. Genes controlling BA absorption, synthesis or receptors are associated with quantitative traits in IBS-D 2. Altered mRNA and protein expression of neurotransmitters, cytokines and ion channels in ileal and colonic mucosa, and BA homeostasis in ileal and colonic mucosa are associated with quantitative in traits in IBS-D; 3. BA sequestration normalizes colonic transit, intestinal permeability, barrier junction proteins and bowel function in IBS-D with increased fecal BA excretion. Significance: These innovative studies will enhance understanding the mechanisms, diagnosis and treatment of BA-associated IBS-D.